Acyl deoxyribonucleoside derivatives and uses thereof

ABSTRACT

The invention relates to compositions comprising acyl derivates of 2′-deoxyribonucleosides. The invention also relates to methods of treating or preventing radiation, mutagen and sunligth-induced cellular damage, methods for improving wound healing and tissue repair, and methods for ameliorating the effects of aging comprising administering the composition of the present invention to an animal.

This application is a Continuation of application Ser. No. 08/153,163,filed Nov. 17, 1993, now U.S. Pat. No. 6,020,320, which is a Divisionalof Ser. No. 07/958,598, filed Oct. 7, 1992, now abandoned, which is aContinuation of Ser. No. 07/533,933, filed Jun. 5, 1990, now abandoned,which is a Continuation of Ser. No. 07/115,923, filed Oct. 28, 1987, nowabandoned, the contents of which are hereby incorporated by reference inthis application.

FIELD OF THE INVENTION

This invention relates generally to acyl derivatives ofdeoxyribonucleosides and to the use of those derivatives to enhance thedelivery of exogenous deoxyribonucleosides to animal tissue. Morespecifically, this invention relates to the acyl derivatives of2′-deoxyadenosine, 2′-deoxyguanosine, 2′-deoxycytidine and2′-deoxythymidine and the use of those novel derivatives to increase thebioavailability of the deoxyribonucleosides to animal tissue and therebyto support cellular metabolic functions. Even more specifically, thisinvention relates to the use of the novel acyl derivatives to treat orprevent a variety of physiological and pathological conditions in celltissue, including damage by radiation, sunlight, mutagens, wounds, andother conditions.

BACKGROUND OF THE INVENTION

There are many physiological and pathological conditions of animaltissue where the supply of exogenous deoxyribonucleosides may haveuseful therapeutic applications. In the treatment of wounds, repair ofliver tissue, promotion of repair and survival after radiation, andnumerous other conditions, the supply of DNA and/or deoxyribonucleosidesat a high and sustained level may substantially improve the natural DNAand tissue repair processes of the affected cells.

In promoting wound healing, liver regeneration, recovery from radiationdamage, and in other pathological and physiological conditions, it islikely that exogenously supplied DNA serves merely as a storage depotfor deoxyribonucleosides. That depot gradually releasesdeoxyribonucleotides and deoxyribonucleosides during enzymaticdegradation. Thus the administration of deoxyribonucleosides orderivatives disclosed herein may have value as a method for deliveringthose deoxyribonucleosides to tissues, which method is preferable to theadministration of foreign DNA insofar as wound healing, tissueregeneration, recovery from irradiation, and the like, is concerned.

A number of investigators have attempted to use DNA and/ordeoxyribonucleosides to treat a variety of conditions in experimentalanimals and to enhance or augment cellular repair processes, includingDNA repair. It has been demonstrated that administration of exogenousDNA to experimental animals after exposure to ionizing radiation canresult in dramatically increased survival and functional recovery.Studies on cell cultures in vitro demonstrate that the actualrestorative agents are probably deoxyribonucleosides, the enzymaticdegradation products of DNA. These compounds enhance the actual repairof damaged DNA in vitro. However, depolymerized DNA ordeoxyribonucleosides administered to animals were ineffective inpromoting survival or recovery after irradiation. Kanazir et al., Bull.Inst. Nuc. Sci “Boris, Kidrinch” 9:145-153 (1959). There is reason tobelieve that this apparent contradiction is due to the rapid catabolismof deoxyribonucleosides in vivo by the liver and other organs. Thus,after administration of deoxyribonucleosides, tissues were only exposedto effective concentrations for a matter of minutes. Beltz, et al.,Bioch. Biophys. Acta 297:258-267 (1973). In cell cultures, optimumsurvival after irradiation was found when deoxyribonucleosides werepresent in the incubation medium for at least 3 hours. When DNA isadministered by intraperitoneal injection, it is gradually depolymerizedto give a sustained release of free deoxyribonucleosides into thecirculation. DNA is not, however, a suitable pharmaceutical agent toadminister to humans, either orally or parenterally.

Hunting, D. J., et al., Carcinogenesis 6:1525-1528 (1985), disclose thatdeoxyribonucleotide synthesis is rate limiting for excision repair ofUV-induced DNA damage. The authors found that there was an increase inrepair ligation in cells made permeable to added deoxyribonucleotidetriphosphates.

Golba, S., et al., Int. J. Rad. Biol. 13:261-268 (1967), disclose thatafter whole-body irradiation, administration of heterologous DNAimproved survival and accelerated the rate of recovery of body weightand of red blood cells, granulocytes and lymphocyte counts in theperipheral blood. No secondary disease or change in the blood count wasobserved in the next 12 months. Goh, K., Proc. Soc. Exp. Biol. Med.145:938-943 (1974), discloses addition of exogenous deoxyribonucleotidesresulted in prevention or healing of “pulverized” chromosomes found incultures of leukocytes taken from a human subjected to accidentalexposure to fast neutron and gamma irradiation. Horikawa, M., et al.,Exp. Cell Res. 34:198-200 (1964), disclose the effect of the addition ofvarious cell extracts and compounds to an incubation medium containingmouse L cells in culture which were irradiated in culture withX-irradiation (2000 R). Homogenates of L cells, L cell nuclei, orpurified DNA from either L cells or salmon sperm all strongly enhancedthe survival of the irradiated cells. RNA from either yeast or L cellswas found to be ineffective. The authors suggest that the DNAhydrolysates (e.g., deoxynucleotides) are the actual reactivatingagents, since heterologous DNA is as effective as homologous DNA.

Pantic, V., et al., Nature 193:993-994 (1962), disclose administrationof DNA to X-irradiated rats given lethal doses of radiation. The authorsfound that while DNA treatment did not totally prevent cellular damagein the intestine and liver after irradiation, tissue structure andfunction were much closer to normal in DNA-treated animals examined 4 or9 days after irradiation than in untreated irradiated controls.

Paoletti, C., et al., Rev. Francais. Etudes Clin. et Bio. 9:950-955(1964), disclose a study on the effect of administration of DNA and2-aminoethyl-isothiouronium (AET) to rats. Mice were given a mixture ofAET and thiogel orally, then irradiated (700 rad) and subsequently giveni.p. injections of 1 mg calf thymus DNA. The mice receiving the DNAinjections recovered their weight and initial leukocyte counts morerapidly than mice similarly treated but not receiving the DNAinjections.

Petrovic, D., et al., Int. J. Radiat. Biol. 18:243-258 (1970), discloseevidence concerning the molecular basis of the restorative effect of DNAin cultured mammalian cells. The authors found that the survival ofirradiated cells in culture was enhanced by the addition of either DNAor equimolar amounts of deoxyribonucleosides. DNA was effective only ifserum containing active deoxyribonuclease was present in the incubationmedium. Thus, the authors concluded that the deoxyribonucleosides wereprobably the actual reactivating factors responsible for repair ofradiation-induced damage. In another study, Petrovic disclosed thatmaximal restoration is attained when deoxyribnucleosides are in theincubation medium for at least 3 hours after irradiation. The bestrestoration was achieved with either a mixture of all four majordeoxyribonucleosides, or a combination of deoxyguanosine with eitherdeoxyadenosine or deoxycytidine. Petrovic, D., et al., Studia Biophysica43:13-18 (1974). Petrovic et al. also report that in irradiated HeLacells, treatment with a mixture of the four major deoxyribonucleosidesincreased survival. Petrovic et al., Int. J. Radiat. Res. 11:609-611(1967).

Savkovic, N., Nature 203:1297-1298 (1964), discloses that 8 or 17 dayold rats subjected to X-radiation (600 rem), and immediately treatedwith homologous testes DNA, had a much higher fertility rate than diduntreated irradiated controls. Histological studies demonstrated thatDNA treatment after irradiation markedly protected the structuralintegrity of the testes and the function of the spermatogenic processes.Savkovic also reported that heterologous DNA extracted from variousorgans of adult rats was effective in enhancing the survival of micesubjected to irradiation. The DNA reduced the effects of radiation by afactor of 9 to 13. Savkovic, N., et al., Nature 211:1179-1180 (1966).Savkovic, N., et al., Int. J. Rad. Biol. 9:361-368 (1965) also disclosethat treatment of irradiated rats with homologous DNA, isolated fromliver, thymus and spleen, increased survival and fertility of thesurvivors. The death rate of the progeny of the irradiated rats wasstrongly reduced in the case of animals that received DNA afterirradiation.

In another study, exposure of cultured calf liver cells to X-radiationwas found to cause chromosomal damage. When cells were incubated witheither DNA or equimolar concentration of deoxyribonucleotides afterirradiation, there was a marked reduction in the incidence of chromosomedamage. A mixture of dAMP and dGMP was as effective as a mixture of allfour major deoxyribonucleotides. Ribonucleotides were ineffective inpreventing radiation-induced chromosome damage. Smets, L. A., et al.,Int. J. Rad. Biol. 13:269-273 (1967).

In a related study, administration of dCMP or dTMP to irradiated micewas found to improve the restoration of hematopoietic function. Soska,J., et al., Folia Biologica 5:190-198 (1959).

In another study of mice irradiated with gamma radiation, administrationof either a yeast RNA hydrolysate, an equimolar mixture of3′-nucleotides or a mixture of nucleosides resulted in a significantprolongation of life span. However, long-term survival was not enhanced.The nucleic acid derivatives were administered 30 minutes, 2 days, and 4days after irradiation. The author observed that the nucleosides,nucleotides, and RNA hydrolysate did not increase the number ofsurviving stem cell colonies in spleen or bone marrow, but ratherappeared to improve the functional capacity of irradiated cells duringthe critical period after irradiation. These compounds also appeared toaccelerate the process of maturation and differentiation of the progenyof surviving stem cells. Sugahara, T., et al., Brookhaven Symposia inBiology, 284-302 (1967).

In a study of guinea pigs subjected to X-radiation, animals given RNA orATP immediately before and after irradiation had much higher 21-daysurvival rates than did untreated irradiated controls. Most of theanimals that survived the 21-day observation period recovered fully,with no secondary radiation-induced disease. Wagner, R., Int. J. Rad.Biol. 12:101-112 (1967).

In another study, administration of DNA from different sources,including calf thymus, rat liver and spleen, herring and salmon sperm,and Ehrlich ascites carcinoma cells was studied in rats given lethaldoses of gamma irradiation. All forms of DNA significantly increased thesurvival of the irradiated rats. The quantitative differences in theeffects of the DNA from different sources were directly related to themolecular weight. The authors found a reduction in therapeuticefficiency which is proportional to the reduction in molecular size uponDNA shearing. Wilczok, T., et al., Int. J. Rad. Biol. 9:201-211 (1965).

The incidence of chromosomal abnormalities in lymphocytes fromradiologists chronically exposed to X-rays, was determined before,during, and after treatment with DNA and ATP. The basal incidence ofchromosomal damage was substantially higher than in unexposed controlsubjects. Daily injection of DNA and ATP resulted in 2 to 3 folddecreases in the frequency of chromosomal abnormalities. Followingdiscontinuation of treatment, the incidence of chromosomal damagereturned toward pretreatment levels. (Goyanes-Villaescusa, Lancet II:575(1973).

There have also been reports on the use of DNA preparations to treatwounds. For example, Dumont, Ann. Surg. 150:799 (1959), disclose thatexogenous DNA, applied to experimental wounds in rabbit ears,accelerated the growth of granulation tissue in the wounds. A mixture ofDNA plus deoxyribonuclease (the enzyme primarily responsible fordegradation of DNA) was more effective in accelerating fibroplasia thaneither DNA or deoxyribonuclease alone. The total amount of granulationtissue formed after treatment with DNA was not greater than in untreatedcontrols; the onset and rate of its growth were however significantlyaccelerated. The authors suggest that low polymer DNA fragments are theactual active agents.

Nicolau et al., Der Hautartzt 17:512 (1966), disclose a study onexperimental skin wounds on the backs of rats which were treated dailywith a It solution of DNA in physiological saline. The wounds treatedwith local application of DNA were cicatrized within four to eight days;those treated only with physiological saline were cicatrized only after10 to 15 days.

Marshak et al., Proc. Soc. Exp. Biol. Med. 58:62 (1945), disclose thatapplication of DNA to experimental skin wounds in rats resulted in asignificant acceleration of the growth of granulation tissue within thewounds, as compared to untreated controls. Although the granulationtissue appeared sooner in treated wounds, the final amount ofgranulation tissue was not abnormal.

Newman et al., Am. J. Physiol. 164:251 (1951), disclose a study of ratssubjected to partial hepatectomy. The course of liver regeneration wasfollowed for 11 days. The livers of rats treated with DNA regeneratedsignificantly faster than livers in untreated animals. RNA treatmentalso accelerated liver regeneration, though not as markedly asDNA-administration.

Certain derivatives of deoxyribonucleosides have been prepared. Casidaet al., Biochemical Pharmacology vol. 15, p. 627-644, 1966, describe thepreparation of the 3′5′-diacetyl, dipropionyl and dibutyryl esters of2′-deoxythymidine. Rosowsky et al., Cancer Treatment Reports vol. 65No.1-2, p. 93-99, January/February 1981, and Ensminger et al.,Biochemical Pharmacology vol. 28, p. 1541-1545, October 1978, describethe use of thymidine 5′-O-pivaloate to supply thymidine to tissues.

Since the primary determinant of recovery or survival after exposure toionizing radiation or chemical mutagens is the preservation or repair ofDNA, a number of compounds have been found which, when present in anorganism at the time of exposure to radiation or chemical mutagens,attenuate the damage to DNA and other cellular structures. Included inthis class of compounds are antioxidants, sulfhydryl compounds, and theenzymes superoxide dismutase and catalase. However, these compounds havebeen found to be only moderately protective or practical to use in vivo,in part because they can be toxic in effective concentrations. Sincethese compounds must be present in the organism at the time of exposureto radiation or chemical mutagens, they are obviously not useful in thecase of unexpected or accidental exposure.

Reportedly, sulfhydryl compounds are the most effective radioprotectiveagents known. Examples of these compounds include mercaptoethylamine(MEA), 2-β-amino-ethyl-isothiouronium-Br—HBr (AET), 5-hydroxytryptamine(HT), and 5-2-(3-amino-propylamino)ethylphosphorothiotoic acid(WR-2721). However, many of these compounds are toxic. Thus, severalinvestigators have attempted to increase protection against radiationdamage and to decrease toxicity by using mixtures of these chemicalprotectors. The results of these studies demonstrate that theadministration of mixtures of radioprotectors not only increases thedegree of protection for short and long term survival compared with thatfrom each substance given separately, but also diminishes the toxicityof compounds such as AET or MEA. Administration of sulfhydryl chemicalradioprotectors before exposure to radiation diminishes markedly thechanges induced by radiation in the structures. Maisin, J. R., in:Symposium on Perspectives in Radioprotection, Armed Forces RadiobiologyResearch Institute, Bethesda, Md., p. 53 (1987).

Thiols reportedly protect DNA by mechanisms comprising hydroxyl radicalscavenging and DNA radical repair mechanisms. Thus, the extent ofinteractions of thiols with DNA determines the amount of protection.Cationic thiols (2-[(aminopropyl)amino]ethanethiol (WR-1065) andcysteamine) are better protectors than neutral thiol (2-mercaptoethanoland dithiothreitol) which are in turn better protectors than anionicthiols (glutathione (GSH), 2-mercaptoethanesulfonic acid, andmercaptosuccinate). Such differential binding provides a basis forunderstanding why WR-1065, which scavenges hydroxyl radicals at a ratecomparable to that for GSH, effectively protects cells at concentrationswell below those of GSH. Fahery, R. C., ibid., p. 31.

In studies of Chinese hamster V-79 cells treated with gamma radiationand with bromodeoxyuridine (BrdUrd) and light photolysis were compared.When treated with gamma radiation, WR-2721 was found to improve cellsurvival both by acting as a reducer of gamma radiation, and by causingincrease in DNA repair and increase in rejoining of DNA strand breaks.Cysteamine has been shown to act as a reducer of gamma radiation damagewithout affecting the rejoining of strand breaks or DNA repair capacity.Nicotinamide (NA) has been shown to directly affect DNA repair throughthe polyADPribose system which is activated by DNA single strand breaks,thus providing NA concentration dependent protection or sensitization.These compounds exhibit a different effect on cells treated with BrdUrdand light compared with gamma radiation. WR-2721 does not reduce strandbreak formation. MEA and NA reduce damage formation by about 30%.WR-2721 did not affect the rejoining of BrdUrd/light-induced DNA strandbreaks. Only NA increased the repair capability of cells subjected toDrdUrd and light damage. Prager, A., et al., ibid., p. 43.

In addition to the use of-thiols, radioprotection has been achieved with“biological response modifiers” (BRM), either alone or in combinationwith other agents. Such biological response modifiers include glucan,OK-432, Biostim, PSK, Lentinan, Schizophyllan, Rhodexman, Levan,Mannozym, and MVE-2. Of these BRM's, glucan was found to be the mostradioprotective. Glucan is a beta 1-3 polyglycan isolated from the yeastSaccharomyces cerevisiae. Glucan's radioprotective capacity isattributed to its ability both to protect and/or enhance recovery ofhemoatopoietic stem cell populations, and to enhance or maintain thefunction of macrophage cell populations important in combattingotherwise lethal post-irradiation opportunistic infections. Thecombination of glucan and WR-2721 resulted in both additive andsynergistic radioprotective effects. Patchen, M. L., ibid., P. 68.

Other polysaccharides have also been found to be radioprotective.Intravenous administration of the polysaccharide extracted from theyeast Rhodotorula rubra, mannane mannozyme (MMZ), and the particulatepolyglucans GLP/B04 and GLP/B05 (unbranched glucans with alternatingB-1,3 and B-1,6 bonds), significantly decreased the mortality of miceexposed to a single dose of X-rays. Maisin, J. R., ibid., p. 69.

The cytokines IL-2 and TNF have also been found to be effectiveradioprotective compounds. Cytokines are released upon administration ofnumerous inflammatory agents. Many of these inflammatory agentsstimulate the reticuloendothelial system and are radioprotective. Neta,R., ibid., p. 71.

Thymic peptides, such as thymic factor TP-5, have also been reported toreverse or greatly ameliorate immune depression due to limited portalirradiation of thymus, circulating blood, and lymphoid tissues. Theimmune restorative effect of thymic factors is due to their maturationaleffect on bone marrow immunocyte precursors. Chretien, P. B., ibid., p.72.

The antioxidant enzymes glutathione peroxidase (GSH-Px), superoxidedismutase (SOD), catalase, glutathione reductase and glutathionetransferase scavenge free radical species produced by radiation and/orthe products of free radical cellular damage, and thus play a role inradioprotection. GSH-Px exhibits and best correlation between enzymeactivity and cell radiosensitivity. Administration of enzymepreparations or drugs or chemicals which mimic or activate or inducethese enzymes may enhance radioprotection. The radioprotectors MEA,WR-2721 and diethyldithiocarbamate (DDC) enhance mouse liver GSH-Pxactivity 1 to 2 hours after administration. Selenium andselenium-containing compounds also exhibit a small radioprotectiveeffect. The levels of GSH-Px in mouse bone marrow were found to increase30% 24 hours after administration of selenium. When selenium wasadministered before WR-2721, a decrease in toxicity and an increase inradioprotection was observed. Superoxide dismutase (SOD) and catalasewere also observed to increase upon administration of selenium. Inaddition, metal ions and metal-containing compounds which mimicantioxidant enzymes may also act as radioprotectors. Copper and zincmetal ions in SOD are marginally radioprotective. Mimetics of SODinclude bis (3,5-diisopropylsalicylato) copper and the bivalent coppercomplex of 3-mercapto-2-hydroxypropylether of dextran. Dumar, K. S.,ibid., p. 89. For a review on SOD, see Fridovich, I., Annu. Rev.Biochem. 44: 147-159 (1975).

Induction of metallothionein (MT) in the body by treatment with someheavy metals or immunostimulants has been found to be a potent means forinducing radioprotection. The metal salts CdCl₂, MnCl₂ or zinc acetateor the immunostimulants OK-432 or IL-1 elevates MT levels in the liverof pretreated mice 10 to 20 times of the control level. The number ofleukocytes as well as erythrocytes were reduced temporarily even inpretreated mice. However, the cell counts of pretreated mice showed afaster recovery. Matsubara, J., et al., ibid., p. 99.

Vitamin A and beta carotene have also been suggested as radioprotectiveagents. They may be involved, in ameliorating the oxidative damage intissues of irradiated mammals which results from production of freeradicals such as hydroxy radicals or H₂O⁺ and its daughter products.Radiation may also create injury to cell structures either by the directeffect of radiation or by the production of toxic metabolites. Radiationinjury results in disturbed extracellular and intracellular oxygenlevels and perturbed intracellular electron transport and metabolism.Oxidative damage may be enhanced by sudden elevations of local oxygenlevels caused by reperfusion of tissues after radiation-inducedvasoconstriction or by reversal of radiation-inducedbronchoconstriction. Damage occurs where local oxygen levels are inexcess of what the tissues can consume. Vitamin A and beta carotene werefound to exhibit protective action in rodents exposed to whole body andlocal radiation. Seifter, E., et al., ibid. p. 104.

Prostaglandins and related compounds of the arachidonic acid cascadeprotect cells in vivo from some degree of ionizing radiation injury.Among the array of physiological actions of prostaglandins is theprotection of cells and tissues from a variety of injuries includingstrong acids, bases, and absolute ethanol. Prostaglandins were found toexhibit maximal protection at levels a thousandfold lower than thoseneeded for WR-2721. Hanson, W. R., ibid., p. 105.

Methylene blue, a compound used clinically as an anti-inflammatory,antimalarial, and antibacterial agent as well as in the treatment ofcarbon monoxide poisoning and as an antidote for cyanide poisoning, asalso found to protect the intestinal mucosa of rats subjected tosublethal radiation-induced damage. Irradiation damages tissues throughthe production of highly bioactive free radical species. Therefore, itwas hypothesized that methylene blue would also protect irradiated ratsfrom free-radical mediated tissue damage. Scheving, L. E., et al.,ibid., p. 115.

In addition, N-arylacetyldehydroalanines reportedly inhibit superoxideanion and hydroxyl free radical-mediated processes, thereby providingradioprotective activity. Buc-Calderone, P., et al., ibid., p. 116.

OBJECTS OF THE INVENTION

While the strategy of delivering DNA and/or deoxyribonucleosides tophysiologically or pathologically damaged tissue has been recognized,the art has heretofore failed to provide satisfactory methods forintroducing deoxyribonucleosides in sufficiently high and reliableamounts to successfully treat the pathological and physiologicalconditions and to promote cellular repair and survival of the animal.Moreover, although a variety of compounds have been developed whichprotect animals against some effects of ionizing radiation or chemicalmutagens, deoxyribonucleosides provided to tissues for a sufficient timehave the greatest clinical potential for post-exposure treatment of suchdamage. Clinical implementation of this strategy, however, awaitsdevelopment of satisfactory and convenient methods for deliveringadequate quantities of deoxyribunucleosides to tissues in vivo.Similarly, full appreciation and clinical implementation of the capacityof deoxyribonucleosides to promote wound healing or tissue repair awaitsdevelopment of satisfactory methods for their delivery to tissues invivo.

It is thus a primary object of this invention to identifypharmaceutically acceptable compounds which can efficiently be used todeliver pharmacologically effective amounts of deoxyribonucleosides ortheir respective derivatives to animal tissue.

It is still a further object of this invention to provide a family ofdeoxyribonucleoside derivatives which can be effectively administeredorally or parenterally, which have no undesirable toxic effects, andwhich can be administered to animals and humans to effectively promotecellular repair in a number of physiological and pathological conditionsand to promote survival of the animal when administered after exposureto radiation has occurred.

It is still a further and related object of this invention to providecertain derivatives of 2′-deoxyadenosine, 2′-deoxyguanosine,2′-deoxycytidine, and 2′-deoxythymidine which, when administered to ananimal, enhance the bioavailability of those deoxyribonucleosides to theanimal tissue.

It is a related object of this invention to substantially improve thebioavailability of 2′-deoxyadenosine, 2′-deoxyguanosine,2′-deoxycytidine, and 2′-deoxythymidine by enhancing the transport ofthese deoxyribonucleosides across the gastrointestinal tract, theblood-brain barrier, and other biological membranes.

It is still a further and more specific object of this invention toprovide a family of deoxyribonucleoside derivatives for the treatment ofa variety of heart, muscle, liver, bone, skin, and other pathologicaland physiological conditions.

It is still a further object of this invention to providedeoxyribonucleoside derivatives and methods for using those derivativeswhich are safe, inexpensive, and which accelerate the normal cellularprocesses of regeneration and healing.

SUMMARY OF THE INVENTION

These and other objects of the invention are achieved by theadministration of certain acyl derivatives of 2′-deoxyadenosine,2′-deoxyguanosine, 2′-deoxycytidine, and 2′-deoxythymidine. These acylderivatives can be used to prevent or treat radiation, sunlight andmutagen-induced cellular damage, to improve the healing of wounds, orrepair damaged tissues, and in the treatment of other physiological andpathological tissue conditions.

Broadly, the acyl derivatives of 2′-deoxyadenosine are those having theformula

wherein R is hydrogen or an acyl radical of a metabolite other thanacetyl, with the proviso that at least one R is not hydrogen, or thepharmaceutically acceptable salt thereof.

The preferred acyl derivatives of 2′-deoxyadenosine are those having theformula

where R is H or an acyl group derived from a carboxylic acid selectedfrom one or more of the group consisting of pyruvic acid, lactic acid,enolpyruvic acid, an amino acid, a fatty acid other than acetic acid,lipoic acid, nicotinic acid, pantothenic acid, succinic acid, fumaricacid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, andcarnitine, with the proviso that at least one R is not hydrogen, or thepharmaceutically acceptable salt thereof.

Broadly, the acyl derivatives of 2′-deoxyguanosine are those having theformula

wherein R is hydrogen or an acyl radical of a metabolite other thanacetyl, with the proviso that at least one R is not hydrogen, or thepharmaceutically acceptable salt thereof.

The preferred acyl derivatives of 2′-deoxyguanosine are those having theformula

wherein R is H or an acyl group derived from a carboxylic acid selectedfrom one or more of the group consisting of pyruvic acid, lactic acid,enolpyruvic acid, an amino acid, a fatty acid other than acetic acid,lipoic acid, nicotinic acid, pantothenic acid, succinic acid, fumaricacid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, andcarnitine, with the proviso that at least one R is not hydrogen, or thepharmaceutically acceptable salt thereof.

Broadly, the acyl derivatives of 2′-deoxycytidine are those having theformula

wherein R is hydrogen or an acyl radical of a metabolite other thanacetyl, with the proviso that at least one R is not hydrogen, or thepharmaceutically acceptable salt thereof.

The preferred acyl derivatives of 2′-deoxycytidine are those having theformula

wherein R is H or an acyl group derived from a carboxylic acid selectedfrom one or more of the group consisting of pyruvic acid, lactic acid,enolpyruvic acid, an amino acid, a fatty acid other than acetic acid,lipoic acid, nicotinic acid, pantothenic acid, succinic acid, fumaricacid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, andcarnitine, with the proviso that at least one R is not hydrogen, or thepharmaceutically acceptable salt thereof.

Broadly, the acyl derivatives of 2′-deoxythymidine are those having theformula

wherein R is hydrogen or an acyl radical of a metabolite other than afatty acid having less than five carbon atoms, with the proviso that atleast one R is not hydrogen, or the pharmaceutically acceptable saltthereof.

The preferred acyl derivatives of 2′-deoxythymidine are those having theformula

wherein R is B or an acyl group derived from a carboxylic acid selectedfrom one or more of the group consisting of pyruvic acid, lactic acid,enolpyruvic acid, an amino acid, a fatty acid containing 5 or morecarbon atoms, lipoic acid, nicotinic acid, pantothenic acid, succinicacid, fumaric acid, p-aminobenzoic acid, betahydroxybutyric acid, oroticacid and carnitine, with the proviso that at least one R substituent isnot hydrogen, or the pharmaceutically acceptable salt thereof.

The acyl derivatives of 2′-deoxythymidine may also be those having theformula

wherein R″ is hydrogen or an acyl radical of a metabolite, with theproviso that the R″ on nitrogen is not hydrogen, or the pharmaceuticallyacceptable salt thereof.

Preferred acyl derivatives of 2′-deoxythymidine are those having theformula

wherein R″ is H or an acyl group derived from a carboxylic acid selectedfrom one or more of the group consisting of pyruvic acid, lactic acid,enolpyruvic acid, an amino acid, a fatty acid, lipoic acid, nicotinicacid, pantothenic acid, succinic acid, fumaric acid, p-aminobenzoicacid, betahydroxybutyric acid, orotic acid, and carnitine, with theproviso that the R″ on nitrogen is not hydrogen, or the pharmaceuticallysalt thereof.

The invention also includes compounds having formulae I-IV wherein theribose moiety is monoacylated at the 3′ or 5′ position with thederivative of a fatty acid and includes 3′,5′ diacylated derivatives ofcompounds I-IV wherein at least one such substituent is derived from afatty acid having 5 or more carbon atoms.

The acyl derivatives of 2′-deoxyadenosine, 2′-deoxyguanosine,2′-deoxycytidine, and 2′-deoxythymidine having formulae I, II, III, andV, desirably are substituted with an acyl derivative of a carboxylicacid having 3-22 carbon atoms.

Where acyl derivatives of any of the compounds of formulae I-V aresubstituted by an acyl group derived from an amino acid, the amino acidis desirably selected from the group consisting of glycine, the L formsof alanine, valine, leucine, isoleucine, phenylalanine, tyrosine,proline, hydroxyproline, serine, threonine, cysteine, cystine,methionine, tryptophan, aspartic acid, glutamic acid, arginine, lysine,histidine, ornithine, and hydroxylysine.

In a preferred embodiment of the invention, a mixture of at least twoacyl derivatives of 2′-deoxyadenosine, 2′-deoxyguanosine,2′-deoxycytidine, and 2′-deoxythymidine is used. Said compositionscontain at least two of the acyl derivatives having the formulae

wherein R′″ is H or an acyl group derived from a carboxylic acidselected from one or more of the group consisting of pyruvic acid,lactic acid, enolpyruvic acid, an amino acid, a fatty acid, lipoic acid,nicotinic acid, pantothenic acid, succinic acid, fumaric acid,p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, andcarnitine, with the proviso that at least one R is not hydrogen, or thepharmaceutically acceptable salt thereof.

Further substantial benefits may be obtained, particularly where thecompositions of the invention are used to ameliorate the effects ofradiation, if a radioprotective compound is included together with oneor more of the acyl deoxyribonucleosides. The radioprotective compoundsmay be those selected from the group consisting of WR-2721, NAC, DDC,cysteamine, 2-mercaptoethanol, mercaptoethylamine dithiothreitol,glutathione, 2-mercaptoethanesulfonic acid, WR-1065, nicotinamine,5-hydroxytryptamine, 2-aminoethyl-isothiouronium-Br—Hbr, glucans,GLP/B04, GLP/B05, OK-432, Biostim, PSK, Lentinan, Schizophyllan,Rhodexman, Levan, Mannozym, MVE-2, MNR, MMZ, IL-1, TNF, thymic factorTF-5, glutathione peroxidase, superoxide dismutase, catalase,glutathione reductase, glutathione transferase, selenium, CdCl₂, MnCl₂,Zn acetate, Vitamin A, beta carotene, prostaglandins, tocopherol,methylene blue and PABA.

The invention is also embodied in pharmaceutical compositions whichcomprise one or more of the novel deoxyribonucleosides together with apharmaceutically acceptable carrier. In addition, known acetylderivatives of the 2′-deoxyadenosine, 2′-deoxyguanosine,2′-deoxycytidine and 2′-deoxythymidine as well as the fatty acidderivatives of thymidine wherein the acyl group contains 3 or 4 carbonatoms may be used alone, in combination with one another or incombination with one or more novel compounds, in pharmaceuticalcompositions of the invention. The composition may further include aradioprotective compound as described. The compositions may be in theform of a liquid, a suspension, a tablet, a dragee, an injectablesolution, a topical solution, or a suppository.

A skin lotion may be advantageously prepared by combining an effectiveamount of one or more of the acyl deoxyribonucleosides of the inventiontogether with a suitable carrier. Such a skin lotion advantageouslycontains from 0.1 to 5 percent by weight of the deoxyribonucleosidesand, if desirable, the radioprotective compound.

The pharmaceutical compositions of the invention can also be embodied inbioerodible microcapsules, the microcapsules desirably being selectedfrom the group consisting of polylactate or lactate-glycolatecopolymers.

It is believed that the delivery of exogenous deoxyribonucleosides tothe tissue of an animal can be effectively achieved by administering tothat animal an effective amount of an acyl derivative of adeoxyribonucleoside of formulae I-V. By enhancing the delivery ofexogenous deoxyribonucleosides, and thereby increasing theirbioavailability, it may be possible to treat physiological orpathological conditions of the tissues of an animal by essentiallysupporting the metabolic functions thereof. Without being bound bytheory, the invention may work, as well, by increasing thebioavailability of nucleoside anabolites e.g. nucleotides ornucleotide-derived cofactors. Administration of the nucleosides per seincreases their bioavailability but, due to rapid catabolism, this maynot result in significant elevation of nucleotide levels; i.e., one doesnot necessarily get an increase in intracellular levels because at lowernucleoside levels there is rapid uptake by the cells whereas at higherlevels there is saturation and the excess is degraded. The invention isbelieved to work by delivering a steady supply of nucleoside at lowlevels.

It is believed that the novel compounds and compositions of theinvention may be used advantageously in methods for treating cardiacinsufficiency, myocardial infarction, the consequences of hypertension,cirrhosis of the liver, diabetes, senescence, adrenal insufficiency, thecomplications of pregnancy, cerebrovascular disorders, senile dementias,Parkinson's disease, demyelinating disorders, cerebellar ataxia, infantrespiratory distress syndrome, and lung disorders, or to enhance bonehealing or muscle performance.

The specific methods where advantages may be achieved using thecompounds and compositions of the invention include treating orpreventing radiation-induced cellular damage, preventingsunlight-induced cellular damage, ameliorating the effects of aging,preventing mutagen-induced cellular damage, healing damaged tissue,healing skin wounds, healing burn tissue, healing diseased or damagedliver tissue, healing heart muscle damaged as a result of myocardialinfarction, treating damaged bone marrow, and enhancing erythropoiesis.In treating all of these conditions, a compound of the invention, withor without additional carriers, radioprotective compounds, and otheradjuvants, are administered to an animal.

The invention essentially enhances the transport of deoxyribonucleosidesacross biological membranes, including the gastrointestinal tract (i.e.,transport from the gut into the bloodstream) and the blood-brainbarrier. The rapid catabolism by nucleoside phosphorylases or nucleosidedeaminases is also substantially prevented.

Administration of the acylated derivatives offers certain advantagesover the nonderivatized compounds. The acyl substituents can be selectedto increase the lipophilicity of the nucleoside, thus improving itstransport from the gastrointestinal tract into the bloodstream. Theacylated derivatives are effective when administered orally. Theacylated derivatives are resistant to catabolism by nucleosidedeaminases and nucleoside phosphorylases in the intestine, liver, otherorgans, and the bloodstream. Thus, administration of the acylatedderivatives of the invention, either orally or parenterally, allowssustained delivery of high levels of deoxyribonucleosides to the tissuesof an animal.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the rates of degradation ofdeoxyribonucleosides in plasma. The following abbreviations were used:

dT=2′-Deoxythymidine

dC=2′-Deoxycytidine

dG=2′-Deoxyguanosine

dA=2′-Deoxyadenosine.

FIG. 2 is a graph illustrating the disappearance of deoxyadenosinederivatives in plasma.

FIG. 3 is a graph illustrating the disappearance of dexoyadenosinederivatives in liver extract.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A “metabolite” is a chemical compound that is formed by, or participatesin, a metabolic reaction. In the context of this application,metabolites include not only acyl substituents known to be synthesizedwithin the human body, but also naturally occurring (but perhapssynthesized rather than extracted) substituents that might be derivedfrom other animal or plant sources. The limiting criteria are that thecompound should be substantially nontoxic and biocompatible, and shouldreadily enter into metabolic pathways in vivo, so as to presentessentially no toxicity during long-term consumption in the dosesproposed. It is preferable that the substituents be metabolized ratherthan excreted intact (or conjugated through detoxification reactions),as concentration of carboxylic acids within the kidney may lead toundesirable excessive acidity. Therefore, carboxylic acids that normallyor easily participate in intermediary, catabolic, or anabolic metabolismare preferred substituents.

The term “pharmaceutically acceptable salts” means salts withpharmaceutically acceptable acid addition salts of thedeoxyribonucleoside derivatives, which include, but are not limited to,sulfuric, hydrochloric, or phosphoric acids.

The term “coadministered” means that at least two of the acylatedderivatives of the invention are administered during a time framewherein the respective periods of pharmacological activity overlap.

“Acyl derivatives” means derivatives of a 2′-deoxyribonucleoside inwhich a substantially nontoxic organic acyl substituent derived from acarboxylic acid is attached to one or more of the free hydroxyl groupsof the ribose moiety of the deoxyribonucleoside with an ester linkageand/or where such a substituent is attached to a primary or secondaryamine in the pyrimidine ring of deoxycytidine or deoxythymidine, or inthe purine ring of deoxyadenosine or deoxyguanosine, with an amidelinkage. Such acyl substituents are derived from carboxylic acids whichinclude, but are not limited to, compounds from the group consisting ofpyruvic acid, lactic acid, enolpyruvic acid, an amino acid, a fattyacid, lipoic acid, nicotinic acid, pantothenic acid, succinic acid,fumaric acid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid,and carnitinic acid. Preferred acyl substituents are compounds which arenormally present in the body, either as dietary constituents or asintermediary metabolites, which are, essentially nontoxic when cleavedfrom the deoxyribonucleoside in vivo.

“Amino acids” include, but are not limited to, glycine, the L forms ofalanine, valine, leucine, isoleucine, phenylalanine, tyrosine, proline,hydroxyproline, serine, threonine, cysteine, cystine, methionine,tryptophan, aspartic acid, glutamic acid, arginine, lysine, histidine,ornithine, hydroxylysine, and other naturally occuring amino acids.

“Fatty acids” are carboxylic acids-having 2-22 carbon atoms. Such fattyacids may be saturated, partially saturated or polyunsaturated.

Preferred acyl derivatives of 2-deoxyribonucleosides for enhancingtransport across biological membranes are those which are morelipophilic than are the parent nucleosides. In general, lipophilic acylnucleoside derivatives have acyl substituents which are nonpolar (asidefrom the carboxylate group). Lipophilic acyl substituents include, butare not limited to, groups derived from acetic acid, lipoic acid, andfatty acids. One of ordinary skill in the art can determine whether aparticular acyl-substituted nucleoside derivative is more lipophilicthan the underivatized nucleoside using standard techniques, i.e.,comparison of the partition coefficients determined in water-octanolmixtures.

Following passage of the acylated nucleoside derivative from thegastrointestinal tract into the bloodstream, across the blood-brainbarrier, or across other biological membranes, the acyl substituents arecleaved by plasma and tissue esterases (or amidases) to give the freenucleosides. The preferred acyl groups of the invention are naturallyoccurring metabolites in the body, or are compounds which readily enterintermediary metabolic pathways. Thus they offer little toxicity whenreleased in vivo by endogenous esterases or amidases.

The rate of removal of the acyl substituents in vivo is a function ofthe specificity of plasma and tissue deacylating enzymes (primarilyesterases or amidases). Fatty acid substituents containing 4 to 8 carbonatoms are cleaved much more rapidly in vivo than are fatty acids witheither more or fewer carbon atoms. Acyl substituents attached to anamine group in the pyrimidine ring of deoxycytidine or deoxythymidine,or the purine ring of deoxyadenosine or deoxyguanosine, with an amidelinkage are cleaved more slowly than are substituents attached tohydroxl groups of ribose with an ester linkage.

It is also possible to prepare acyl nucleoside derivatives which containboth polar and nonpolar acyl substituents. The polar acyl group willretard passage of the nucleoside derivative from the gastrointestinaltract, allowing for a more sustained delivery of the compound into thebloodstream after a single dose. The polar group may be cleaved byesterases, amidases, or peptidases present in the intestinal tract togive a nucleoside with a nonpolar acyl substituent which may thenefficiently enter the circulation. Polar acyl substituents may be chosenby one of ordinary skill in the art, without undue experimentation,which are cleaved at a slower rate than are nonpolar acyl substituents.

The acyl derivatives are also less susceptible to degradation of thenucleoside moiety by enzymes in plasma and non-target tissues, and arealso less susceptible to elimination from the bloodstream via thekidneys. For parenteral injection, acyl derivatives with polarsubstituents, which are therefore water soluble yet resistant topremature degradation or elimination, may be used with advantage.Preferred acyl substituents for such applications include those derivedfrom glycolic and lactic acids and from amino acids with polar sidechains.

Therapeutic Uses

The lipophilic acyl deoxyribonucleoside derivatives of the invention areuseful for enhancing the transport of the deoxyribonucleosides acrossbiological membranes including the gastrointestinal tract andblood-brain barrier in animals and thereby increase the bioavailabilityof the deoxyribonucleosides. Foremost among such animals are humans;however, the invention is not intended to be so limiting, it beingwithin the contemplation of the invention to treat all animals which mayexperience a beneficial effect from the administration of the acyldeoxyribonucleosides of the invention.

The compositions of the present invention may be administered to ananimal either before or after exposure to radiation, sunlight ormutagens. The acyl derivative form of the deoxyribonucleosides providesan orally effective means for delivery of deoxyribonucleosides totissues. These derivatives may also be given parenterally.Administration of the derivatives avoids the problem of rapid catabolismby gastrointestinal, liver and plasma enzymes.

As shown in FIG. 1, free deoxyguanosine (dG) and deoxyadenosine (dA) aredegraded in plasma at a much higher rate than deoxythymidine (dT) anddeoxycytidine (dC). Thus, the compositions of the invention may includelevels of dG and dA which are higher than the levels of dT and dC tocompensate for this differential rate of degradation.

Acyl substituents for derivatization of dA and dG may be selected whichare slowly cleaved by esterases so that a prolonged bioavailability ofthese deoxyribonucleosides may be maintained. Since dT and dC are moreslowly degraded, an acyl substituent which is more rapidly cleaved maybe used.

The fates of deoxyadenosine, N4-acetyldeoxyadenosine, andN4-valeryldeoxyadenosine in plasma are shown in FIG. 2. Each of thesecompounds was added to separate aliquots of rat plasma, at initialconcentrations of 20 micromolar. The plasma was sampled at various timepoints, and the desired compounds were assayed by liquid chromatography.

Deoxyadenosine (dA) is very rapidly degraded in plasma, disappearingwithin 10 minutes. Administration of this compound to an animal or humansubject would make deoxyadenosine available to tissues for a very shortperiod of time.

N4-acetyldeoxyadenosine and N4-valeryldeoxyadenosine are, however,deacylated in plasma (to form deoxyadenosine) over a period of severalhours. Therefore, administration of either of these compounds wouldresult in prolonged availability of deoxyadenosine to tissues.

The fates of deoxyadenosine, N4-acetyldeoxyadenosine, andN4-valeryldeoxyadenosine in liver extract are shown in FIG. 3. Each ofthese compounds was added to separate aliquots of an aqueous extract ofrat liver, at initial concentrations of 20 micromolar. The extract wassampled at various time points, and the desired compounds were assayedby liquid chromatography.

Deoxyadenosine (dA) is extremely rapidly degraded in plasma,disappearing within 1 minute. The initial degradation product isdeoxyinosine, which is not directly reutilizable by tissues.Administration of deoxyadenosine per se to an animal or human subjectwould make deoxyadenosine available to tissues for only a very shortperiod of time.

N4-acetyldeoxyadenosine and N4-valeryldeoxyadenosine are, however,deacylated in liver extract (to form deoxyadenosine) over a period ofmore than 1 hour. Therefore, administration of either of these compoundswould result in prolonged availability of deoxyadenosine to liver orother organs.

Thus a mixture of several different acyl derivatives of eachdeoxyribonucleoside in an administered dose may be selected to provideoptimal bioavailability. A composition containing3′,5′-diacetyl-2′-deoxycytidine, 5′-palmitoyl-2′-deoxycytidine, andN⁴-palmitoyl-2′-deoxycytidine (and corresponding derivatives of otherdeoxyribonucleosides) provides a more prolonged bioavailability ofnucleosides after a single dose than does an administration of a singleacyl derivative of each nucleoside. Thus, after administration of themixture described above, the acetylated compound is relatively rapidlydeacetylated yielding free deoxycytidine (or other desireddeoxyribonucleosides) shortly after administration. The 5′-palmitoylderivative is deacylated more slowly, providing additional freedeoxycytidine after the deoxycytidine derived from3′,5′-diacetyl-2′-deoxycytidine has been metabolized by tissues. TheN⁴-palmitoyl derivative is deacylated still more slowly than is5′-palmitoyl-2′-deoxycytidine.

The acyl deoxyribonucleoside compositions of the invention also find usein treatment of ultraviolet light-induced cellular damage that occursupon exposure to sunlight. Within 72 hours after exposure to sunlight,there is an increase in the number of epidermal cells and a high rate ofmitotic activity. The rate of cell proliferation decreases after 7 to 10days and the thickness of the epidermis gradually returns to normalwithin the next 30 to 60 days. Damage to DNA by sunburn-producing UVlight (290-320 nm) may result in mutation or cell death. The principalphotoproducts are pyrimidine dimers (e.g., thymine dimers). Cellmembranes, DNA, RNA, protein and other molecules may be altered, and thesynthesis of DNA, RNA and protein may be temporarily inhibitedimmediately after irradiation. New synthesis is evident by 24 hours andis maximal by 60 hours. Harrison's Principles of Internal Medicine,Petersdorf et al., eds., 10th Edition, McGraw-Hill Book Company, NewYork, N.Y., p. 276 (1983). Thus, administration of the acyldeoxyribonucleosides of the invention provides optimal levels ofdeoxyribonucleosides during the period of DNA repair after sunlightexposure to ensure maximal cellular protection and repair.

The acyl deoxyribonucleoside composition may be formulated as part of asuntan lotion that may be applied before or after exposure to sunlight.The suntan lotion may also comprise one or more sun blockers such asPABA, esters of PABA, and other non-PABA chemical sunscreens. SeeHarrison, supra, p. 279. The acyl deoxynucleotides are absorbed by theskin and taken up by cells. The acyl deoxyribonucleosides are thencleaved by tissue esterases to give free deoxyribonucleosides in amountseffective for repair of sunlight-induced chromosomal damage. Thecombination of the acyl deoxyribonucleoside compositions and a sunblocker such as PABA offers maximal protection of the skin from the sun.

The acyl deoxyribonucleoside compositions of the invention also find usein ameliorating the effects of aging by providing a high and sustainedlevel of deoxyribonucleosides to enhance the natural DNA repairprocesses of cells, and thereby, treating the naturally occurringprogressive accumulation of damage to DNA which occurs on aging.Compositions for treatment or amelioration of the effects of aging maybe applied topically, in the form of a skin lotion, or may beadministered orally or parenterally.

There are conditions other than radiation damage in which exogenousdeoxyribonucleosides or derivatives thereof have useful therapeuticapplications. Deoxyribonucleic acid has been used to accelerate woundcicatrization or healing, and also to accelerate liver regeneration inexperimental animals. It is likely that in these situations, as well asin the situations where DNA is used to promote survival afterirradiation of animals, the DNA is serving as a storage depot fordeoxyribonucleosides, which gradually releases the deoxyribonucleotidesand deoxyribonucleosides during enzymatic degradation.

Administration of acylated deoxyribonucleosides, as described herein, isa method for delivering deoxyribonucleosides to tissues which ispreferable to the administration of foreign DNA for the purpose ofimproving wound healing or tissue regeneration. Unlike DNA, acylateddeoxyribonucleosides are effective after oral administration; they arealso nonantigenic and are much easier to purify than DNA.

The composition of the present invention may also be administered toenhance the healing of damaged tissue. Such damaged tissue includes skinwounds (e.g., punctures, lacerations, abrasions, etc.), burned tissue(skin, etc.), diseased or damaged liver (from surgery or other wounds ofthe liver, or from cirrhosis or diabetes, etc.), damaged heart muscle(e.g., from myocardial infarction), and damaged bone marrow (e.g., afterradiation treatment or chemotherapeutic treatment).

For the purpose of treating-skin wounds or burns, the compositions maybe applied topically as part of a skin lotion or cream, or as part of abioerodible polymer.

Preferred acyl substituent groups of 2′-deoxyadenosine,2′-deoxyguanosine, 2′-deoxycytidine, and 2′-deoxythymidine are thosewhich form the acetate, valerate and palmitate esters.

Preferred deoxyadenosine derivatives compriseN⁶-palmitoyl-3′,5′-diacetyl-2′-deoxyadenosine,N6-palmitoyl-5′-valeryl-2′-deoxyadenosine, and5′-valeryl-2′-deoxyadenosine.

Preferred deoxyguanosine derivatives compriseN²-palmitoyl-3′,5′-diacetyl-2′-deoxyguanosine, and5′-palmitoyl-2′-deoxyadenosine.

Preferred deoxycytidine derivatives compriseN⁴-palmitoyl-2′-deoxycytidine, 5′-palmitoyl-2′-deoxycytidine,N⁴-palmitoyl-3′,5′-diacetyl-2′-deoxycytidine, and3′,5′-dipalmitoyl-2′-deoxycytidine.

Preferred deoxythymidine derivatives comprise5′-palmitoyl-2-deoxythymidine andN³-palmitoyl-3′,5′-diacetyl-2-deoxythymidine.

Compositions within the scope of the invention include those whichcontain mixtures of the acyl derivatives of the deoxyribonucleosides inamounts effective to achieve its intended purpose. Such compositions maycontain 0 to 50 mole percent of the acyl derivative of deoxycytidine, 0to 50 mole percent of the acyl derivative of deoxyguanosine, 0 to 50mole percent of the acyl derivative of deoxythymidine and 0 to 50 molepercent of the acyl derivative of deoxyadenosine, with the proviso thatthe total content of the acyl deoxyribonucleosides adds up to 100 molepercent.

A preferred composition contains 25 mole percent of the acyl derivativeof deoxycytidine, 25 mole percent of the acyl derivative ofdeoxyguanosine, 25 mole percent of the acyl derivative ofdeoxythymidine, and 25 mole percent of the acyl derivative ofdeoxyadenosine.

For treatment of radiation-induced cellular damage, sunburn, or toenhance wound healing, preferred dosages include amounts of the acylderivatives equivalent to 10 to 1000 mg of 2′-deoxyadenosine, 10 to 1000mg of 2′-deoxyguanosine, 10 to 1000 mg of 2′-deoxycytidine and 10 to1000 mg of 2′-deoxythymidine. For example, the composition may comprise13-1330 mg of 3′,5′-diacetyl-2′-deoxyadenosine, 13-1310 mg of3′,3′-diacetyl-2′-deoxyguanosine, 14-1370 mg of3′,5′-diacetyl-2′-deoxycytidine and 14-1350 mg of3′,5′-diacetyl-2′-deoxythymidine. As is understood in the art, incalculating such dosages, the equivalent amount of the2′-deoxyribnucleoside alone is considered, i.e., the acyl substituentand acid addition portion of any pharmaceutically acceptable salt arenot included in the calculation.

For a suntan lotion, 0.1 to 5% by weight of the above compositions maybe added. Generally, for this purpose, the acyl derivative will be inthe form of the free acyl deoxyribonucleosides and not as thepharmaceutically acceptable salts.

Methods of Preparation

When the acid source of the desired acyl derivative has groups whichinterfere with the acylation reactions, e.g., hydroxyl or amino groups,these groups may be blocked with protecting groups, e.g.,t-butyldimethylsilyl ethers or t-BOC groups, respectively, beforepreparation of the anhydride. For example, lactic acid may be convertedto 2-t-butyldimethylsiloxypropionic acid witht-butyldimethylchlorosilane, followed by hydrolysis of the resultingsilyl ester with aqueous base. The anhydride may be formed by reactingthe protected acid with DCC. With amino acids, the N-t-BOC derivativemay be prepared, using standard techniques, which is then converted tothe anhydride with DCC. With acids containing more than one carboxylategroup (e.g., succinic, fumaric, or adipic acid) the acid anhydride ofthe desired dicarboxylic acid is reacted with a 2′-deoxyribonucleosidein pyridine.

3′,5′-Diacyldeoxythymidine may be prepared according to methodsdisclosed by Nishizawa et al., Biochem. Pharmacol. 14:1605 (1965), bytreating deoxythymidine with 2.1 equivalents of an acid anhydride of thedesired acyl compound in pyridine followed by heating to 80-85° C. forat least one hour. Alternatively, deoxythymidine may be treated with 2.1equivalents of an acid chloride in pyridine at room temperature (seeExample 1).

The 5′-hydroxyl group of deoxythymidine may be selectively acylated with1 equivalent of the acid anhydride of the desired acyl compound inpyridine, which is heated to 80-85° C., according to Nishazawa, et al.Alternatively, the acid chloride (1 equivalent) may be reacted withdeoxythymidine in pyridine and DMF at room temperature according toBaker et al., J. Med. Chem. 21:1218 (1978). (See Example 2.)

The 3′-hydroxyl group of deoxythymidine may be selectively acylated byselectively forming the 5′-O-t-butyldimethylsilyl derivative with 1.2equivalents of t-butyldimethylchlorosilane in DMF containing imidizole,followed by acylation of the 3′-hydroxyl group with the appropriate acidanhydride, and cleavage of the 5′-t-butyldimethyl silyl ether accordingto Baker et al. (See Example 3.)

3′,5-Diacyldeoxycytidine may be prepared according to a method adaptedfrom Gish et al., J. Med. Chem. 14:1159 (1971), by treatingdeoxycytidine hydrochloride with 2.1 equivalents of the appropriate acidchloride in DMF. (See Example 5.)

The 5′-hydroxy group of deoxycytidine may be selectively acylated bytreating deoxycytidine hydrochloride with 1.1 equivalents of theappropriate acid anhydride in DMF. Gish et al. (See Example 6.)

The N⁴-amino group of deoxycytidine may be selectively acylated bytreating deoxycytidine with 1.5 equivalents of the appropriate acidanhydride in pyridine which may contain DMF. Alternatively,deoxycytidine may be treated with 1.5 equivalents of the desired acidanhydride in pyridine and DMF. Another procedure for the selectiveacylation of the N⁴-amino group of deoxycytidine involves treatment withan excess of the appropriate acid anhydride in a mixture of water and awater-miscible solvent. (See Example 7.)

3′,5′,N⁴-Triacyl-2′-deoxycytidine, where all the acyl groups are thesame, may be prepared by treating 2′-deoxycytidine with at least 3equivalents of an acid chloride or anhydride derived from the desiredacyl group, in pyridine. To prepare the triacyl derivatives ofdeoxycytidine where the N⁴-acyl group is different from the 3′,5′-acylgroups, the desired N⁴-amino group may be first selectively acylated asdescribed above, followed by acylation of the 3′ and 5′-hydroxy groupswith the desired acid anhydride. Alternatively, the 3′,5′-diacylderivative may be prepared first, followed by acylation of the N⁴-aminogroup. (See Example 8.)

The 3′,5′-diacyl derivative of deoxyadenosine may be prepared bytreatment with 2.1 equivalents of the appropriate acid chloride in DMF.(Adapted from Gish et al., see Example 9.)

The 5′-hydroxyl group of deoxyadenosine may be selectively acylated bytreatment of deoxyadenosine hydrochloride with 1.1 equivalents of thedesired acid chloride in DMF. (Adapted from Gish et al., see Example10.)

The N⁶-amino group of deoxyadenosine may be selectively acylated bytreating deoxyadenosine with the desired acid chloride (1.5 equivalents)in a mixture of pyridine and DMF. Alternatively, deoxyadenosine may betreated with 1.5 equivalents of the desired acid anhydride in pyridineand DMP (adapted from Sasaki et al., Chem. Pharm. Bull. 15:894 (1967)).The N⁶-amino group may also be selectively acylated by treatment ofdeoxyadenosine with 2 equivalents of an appropriate acid anhydride in amixture comprising water and a water-miscible solvent (adapted fromAkiyama et al., Chem. Pharm. Bull. 26:981 (1978)) (See Example 11.)

The 3′,5′-hydroxyl groups and the N⁶-amino group may all be acylated bytreatment of deoxyadenosine with at least 4 equivalents of theappropriate acid chloride or acid anhydride in pyridine. (See Example12.)

Alternatively, the N6-amino group of deoxyadenosine may be firstselectively acylated as described above, followed by acylation of the 3′and 5′ hydroxy groups. Another approach comprises selective acylation ofthe 3′ and 5′-hydroxy groups of deoxyadenosine as described above,followed by acylation of the N⁶-amino group. These methods give triacylderivatives where the N⁶-acyl group is different from the 3′ and 5′ acylgroups.

3,5-Diacyl-2′-deoxyguanosine may be prepared by treating deoxyguanosinehydrochloride with 2.1 equivalents of the appropriate acid chloride inDMF. (Adapted from Gish et al., see Example 13.)

The 5′-hydroxyl group of deoxyguanosine may be selectively acylated bytreatment of deoxyguanosine hydrochloride with 1.1 equivalents of theappropriate acid chloride in DMF. (Adapted from Gish et al., see Example14.)

The N²-amine of the purine ring of deoxyguanosine may be selectivelyacylated by treating deoxyguanosine with 1.5 equivalents of theappropriate acid anhydride in pyridine or a mixture of pyridine and DMF(adapted from Sasaki et al., Chem. Pharm. Bull. 15:894 (1967).Alternatively, deoxyguanosine may be treated with a twofold excess ofthe appropriate acid anhydride in a mixture of water and awater-miscible solvent (adapted from Akiyama et al., see Example 15).

3′,5′,N²-triacyldeoxyguanosine, where all the acyl groups are the same,may be prepared by treating deoxyguanosine with 4 equivalents of theappropriate acid chloride or acid anhydride in pyridine. (See Example16.)

To prepare the triacyl derivative of deoxyguanosine where the N²-acylgroup is different than the 3′ and 5′ acyl groups, the N²-acyl group maybe selectively acylated as described above, followed by acylation of the3′ and 5′ hydroxyl groups. Alternatively, the 3′ and 5′ hydroxyl groupsmay be acylated first, followed by acylation of the N²-amino group.

These acyl compositions may be administered chronically to an animalwhich is at risk of either exposure to radiation, sunlight or chemicalmutagens. The acyl compositions of the invention may also beadministered after exposure to radiation, sunlight or chemical mutagensor after a wound is inflicted to enhance the repair of DNA and therebyto ameliorate the damage and promote survival of the animal.Advantageously, the compositions of the invention may be administeredbefore or after radiotherapy or chemotherapy to ameliorate undesiredside effects of the treatment.

The acyl compositions of the invention may also be coadministered withother radioprotective compounds such as WR-2721, NAC, DDC, cysteamine, 2mercaptoethanol, mercaptoethylamine, dithiothreitol, glutathione,2-mercaptoethanesulfonic acid, WR-1065, nicotinamide,5-hydroxytryptamine, 2-2-aminoethyl-isothiouronium-Br—Hbr, glucans,GLP/BO4, GLP/BO5, OK-432, Biostim, PSK, Lentinan, Schizophyllan,Rhodexman, Levan, Mannozym, MVE-2, MNR, MMZ, IL-2, TNF, thymic factorTF-5, glutathione peroxidase, superoxide dismutase, catalase,glutathione reductase, glutathione transferase, selenium, CdCl₂, MrCl₂,Zn acetate, vitamin A, beta carotene, prostaglandins, tocopherol, andmethylene blue. The administration of these protective compounds alongwith the acyl derivatives of the invention provides protection greaterthan if the acyl derivatives or the other agents were given alone.

Mercaptoethylamine (cysteamine) may be prepared according to Gabriel,L., Ber. 31:2837 (1898); Knorr, R., Ber. 36:1281 (1903); Mills et al.,J. Am. Chem. Soc. 62:1173 (1940), and Wenker et al., J. Am. Chem. Soc.62:1173 (1940). AET may be prepared according to Clinton et al., J. Am.Chem. Soc. 70:950 (1948); Funahashi, M., J. Agric. Chem. Soc. Japan27:775 (1953); Chemical Abstr. 49:157376 (1955) and Doherty et al., J.Am. Chem. Soc. 79:5667 (1957). 5-Hydroxytryptamine can be preparedaccording to Specter et al., J. Am. Chem. Soc. 73:5514 (1951); Hamlin,U.S. Pat. No. 2,715,129 (1955); U.S. Pat. No. 2,947,757 (1960). For areview, see Erspamer in E. Jucker, Proqress in Drug Research 3:151-367(1961). WR-2721 and WR-1065 may be obtained from the Walter Reed ArmyMedical Center, Washington, D.C. For a synthesis of 2-mercaptoethanol,see Woodward, J. Chem. Soc. 1892 (1948); Peppel et al., U.S. Pat. No.2,401,665 (1964); U.S. Pat. No. 3,394,192 (1968). Dithiothreitol may beprepared according to Evans et al., J. Chem. Soc. 253 (1949).Glutathione can be prepared according to du Vigneaud et al., Biochem.Prepar. 2:87 (1952); Goldschmidt et al., Ber. 97:2434 (1964); and Ozawaet al., Bull. Chem. Soc. Japan 53:2592 (1980). 2-Mercaptoethanesulfonicacid sodium salt may be prepared according to Schramm et al., J. Am.Chem. Soc. 77:6231 (1955); Reppe et al., Ann. 601:111 (1956); U.S. Pat.No. 3,567,835 (1971). Nicotinamide may be prepared according to Truchanet al., U.S. Pat. No. 2,993,051 (1961); Gasson et al., U.S. Pat. No.2,904,552 (1959). Glucans may be isolated from Saccharomyces cerevisiaeaccording to Woods et al., Sci 142:178 (1978). OK-432 may be isolatedfrom Saito et al., Infec. Immun 26:779 (1979). PSK may be isolated fromCoriolus versicolor according to Otsuka, S., et al., Japanese Patent No.7,308,489 (1973). Chemical Abstr. 80:410259g (1974). Lentinan may beprepared according to Abe et al., Gann 74:273 (1983). Schizophyllan maybe prepared according to Saito et al., Infec. Immun 26:779 (1979).Rhodexman may be prepared according to Elinov, Int. J. Immun. 4:265(1982). Mannozym may be prepared according to Nagy et al., Gen. Pharm.12:A31 (1981). Polymannae Rhoderamine may be extracted from Rhodotorularubra according to Komov, Vopr. Med. Rhim. 21:351 (1975).

Cytokines IL-2 and TNF may be prepared according to Godard et al., J.Imuno. Meth 70:233 (1984), and Brown, Photo Sci En. 22:22 (1978),respectively. Thymic peptides may be isolated according to Tomazic etal., Proc. Am. Ass. Cancer Res. 24:196 (1983). Glutathione peroxidasemay be isolated according to Aisaka et al., Agro Biol. Chem. 47:1107(1983). Superoxide dismutase can be prepared according to Bio/Technology5:363-365 (1987). Catalase may be isolated according to Lolli, U.S. Pat.No. 2,703,779; Schroeder et al., Biochim. Biohpys. Acta. 58:611 (1962);Dan, U.S. Pat. No. 2,992,167 (1961); Faucett et al., U.S. Pat. No.3,102,081 (1963). Glutathione reductase may be isolated according toMuramatsu et al., Jap. Soc. Sci. Fisheries 46:757 (1980). Glutathionetransferase may be isolated according to Burgess et al., Fed. Proc.41:1738 (1982). Bis-(3,5-diisopropylsalicylato) copper may be preparedaccording to Sorenson, J. Med. Chem. 27:1747 (1984).3-Mercapto-2-hydroxypropyl ether of dextran may be prepared according toWieczorek, Arch. Immunol. Ther. Exp. (Warsz) 31:715 (1983). Theimmunostimulant IL-1 may be prepared according to Krueger et al., Fed.Proc. 42:356 (1983). Vitamin A may be extracted from fish liver oilaccording to Karrer et al., Helv. Chim. Acta. 16:625 (1933); Heilvon etal., Biochem. J. 26:1178, 1194 (1932). Beta carotene may be isolatedfrom carrots according to Willstatter et al., Z. Physiol. Chem. 64:47(1910); Kuhn et al., Ber. 64:1349 (1931); Barnett et al., U.S. Pat. No.2,848,508 (1958). For preparations of prostaglandins E₁, E₂, F₂, I₂ andX see The Merck Index, Tenth Edition, Windholz, J., et al. (eds.), Merckand Col., Inc., Rahway, N.J., pp. 7777-7781 (1983). For a preparation ofmethylene blue see Fierz-David, H. E., et al., Fundamental Processes ofDye Chemistry, Interscience, New York, p. 311 (1949).N-arylacetyldehydroalanines may be prepared according to Viehe, H. G.,et al., Patent No. WO/02523.

The pharmacologically active acyl derivatives may be combined withsuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds. Thesecan be administered as tablets, dragees, capsules, and suppositories.The compositions can be administered orally, rectally, vaginally, orreleased through the buccal pouch of the mouth, and may be applied insolution form by injection, orally or by topical administration. Thecompositions may contain about from 0.1 to 99%, preferably from about 50to 90% of the active compounds, together with the excipient(s).

The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral usecan be obtained by combining the active compounds with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

Suitable excipients include fillers such as sugars, for example lactose,sucrose, mannitol or sorbitol, cellulose preparations and/or calciumphosphates, for example tricalcium phosphate or calcium hydrogenphosphate, as well as binders such as starch paste, using, for example,maize starch, wheat starch, rice starch or potato starch, gelatin,tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodiumcarboxymethyl cellulose, and/or polyvinyl pyrrolidone.

If desired, disintegrating agents may be added such as theabove-mentioned starches and also carboxymethylstarch, cross-linkedpolyvinyl pyrrolidone, agar, or algenic acid or a salt thereof, such assodium alginate.

Auxiliaries are, above all, flow-regulating agents and lubricants, forexample, silica, talc, steric acid or salts thereof, such as magnesiumstearate or calcium stearate, and/or polyethylene glycol. Dragee coresare provided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated sugar solutions may beused, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquersolutions and suitable organic solvents or solvent mixtures. In order toproduce coatings resistant to gastric juices, solutions of suitablecellulose preparations such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate are used. Dye stuffs or pigmentsmay be added to the tablets or dragee coatings, for example, foridentification or in order to characterize different combinations ofcompound doses.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft-sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules contain the active compound(s) in the form of granules whichmay be mixed with fillers such as lactose, binders such as starchesand/or lubricants such as talc or magnesium stearate, and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids such as fatty oils, liquidparaffin, or polyethylene glycols. In addition, stabilizers may beadded.

Possible pharmaceutical preparations which can be used rectally include,for example, suppositories which consist of a combination of activecompounds with a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, paraffin hydrocarbons,polyethylene glycols or higher alkanols. In addition, it is alsopossible to use gelatin rectal capsules which consist of a combinationof the active compounds with a base. Possible base materials include,for example, liquid triglycerides, polyethylene glycols, or paraffinhydrocarbons.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water soluble form, for example,water soluble salts. In addition, suspensions of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may include substanceswhich increase the viscosity of the suspension which include, forexample, sodium carboxymethylcellulose, sorbitol, and/or dextran.Optionally, the suspension may also contain stabilizers.

The acyl deoxyribonucleosides may be formulated as part of a skin lotionor suntan lotion for topical administration. Suitable formulations fortopical administration include appropriate oily suspensions orsolutions. Suitable lipophilic solvents or vehicles include fatty oils,for example sesame oil or coconut oil, or synthetic fatty acid esters,for example ethyl oleate or triglycerides. These topical formulationsmay be used to treat damaged tissue such as skin wounds or burns, or totreat or prevent sunlight induced cellular damage (sunburn).

For purposes of enhancing wound healing, the compositions of the presentinvention may be formulated as part of bioerodible microcapsules fortopical administration. Such microcapsules may comprise, for example,polylactate or lactate-glycolate copolymers. See Weise, D. L. et al.,Drug Carriers in Biology and Medicine, Gregoriadis, G. et al., AcademicPress, NY p. 237-270 (1979).

The following examples are illustrative, but not limiting of the methodsand compositions of the present invention. Other suitable modificationsand adaptations of a variety of conditions and parameters normallyencountered in clinical therapy which are obvious to the those skilledin the art are within the spirit and scope of this invention.

EXAMPLES OF METHODS TO PREPARE COMPOUNDS OF THE INVENTION Example 1Preparation of 3′,5′-Diacyl-2′-deoxythymidine From Acid Anhydrides

2′-Deoxythymidine is dissolved in anhydrous pyridine at roomtemperature. 2.1 molar equivalents of the acid anhydride of the desiredacyl compound (e.g., acetic anhydride, lactate anhydride, butyricanhydride, etc.) is then added. The reaction mixture is then heated to80-85°C. for 1 to 4 hours, cooled, poured into ice water, and the estersrecovered by extraction with chloroform or a similar solvent. Thechloroform is then washed with ice-cold 0.01 N sulfuric acid, 1% aqueoussodium bicarbonate, and finally water. After drying with sodium sulfate,the chloroform is evaporated and the residual oil or crystals aresubjected to chromatography (adapted from Nishizawa et al., Biochem.Pharmacol. 14:1605 (1965)).

From Acid Chlorides:

To 2′-deoxythymidine dissolved in anhydrous pyridine is added, at 5° C.,2.1 molar equivalents of the acid chloride of the desired acyl compound(e.g., palmitoyl chloride, acetyl chloride, etc.). The mixture is heldat room temperature overnight, added to ice water, and worked up asindicated above (adapted from Nishizawa).

Example 2 Preparation of 5′-Acyl-2′-deoxythymidine

To 2′-deoxythymidine dissolved in anhydrous pyridine is added, at roomtemperature, 1.0 molar equivalent of the acid anhydride of the desiredacyl compound. The reaction is then heated to approximately 80-85° C.for several hours, cooled, poured into ice water, and the estersrecovered by extraction with chloroform or a similar solvent. Thechloroform is then washed in ice-cold 0.01 N sulfuric acid, 1% aqueoussodium bicarbonate, and finally water. After drying with sodium sulfate,the chloroform is evaporated and the residual oil or crystals aresubjected to chromatography. The major product, which is isolated bychromatography is the 5′ substituted ester (adapted from Nishizawa etal.

Alternatively, selectively 5′ acylation of deoxythymidine may beaccomplished by suspending 2′-deoxythymidine in a mixture of pyridineand N,N-dimethylformamide cooled to 0° C. in an ice bath. 1.0 molarequivalent of the acid chloride of the desired acyl compound is addeddropwise to the mixture, which is stirred at 9° C. for 12-24 hours.Water is then added to stop the reaction, and then the solvents areevaporated in vacuo at 50° C. The residue is dissolved in methanol andpurified by chromatography on silica gel (adapted from Baker et al., J.Med. Chem. 21:1218 (1978).

Example 3 Preparation of 3′-Acyl-2′-deoxythymidine

To a stirred suspension of 2′-deoxythymidine in dryN,N-dimethylformamide is added 2.4 molar equivalents of imidazolefollowed by 1.2 molar equivalents of t-butyldimethylchlorosilane. Themixture is stirred with protection from moisture at room temperature for20 hours, at which time the solvent is removed at 50° C. in vacuo. Theresidue is dissolved in 15 ml of ethyl acetate, washed, and evaporatedto give a syrup from which is obtained, by crystallization from hotchloroform by the addition of hexane to the point of opalescence,5′-(t-butyldimethylsilyl)-2′-deoxythymidine.

To a stirred suspension of 5′-(t-butylmethylsilyl)-2′-deoxythymidine indry pyridine cooled to 0° C. is added 1.1 molar equivalents of theappropriate acid anhydride of the desired acyl compound, and the mixtureis stirred with protection from moisture for 20 hours at 0-5° C., atwhich time the reaction is terminated by addition of a few ml of water.The solvent is evaporated and the residue is extracted and evaporated togive a thick, clear syrup, which is then dried in vacuo at 25° C.

The t-butylmethylsilyl group is removed with glacial acetic acid andtetrabutylammonium fluoride in tetrahydrofuran, yielding the desired3′-acyl-2′-deoxythymidine derivative (adapted from Baker et al.

Example 4 Preparation of N³-Acyl-2′-deoxythymidine

The acylation of the secondary amine in the 3 position of the pyrimidinering is accomplished by reacting 3′,5′-diacyldeoxythymidine with 1.1molar equivalents of the acid chloride of the desired acyl substituentin an aprotic solvent (such as ether, dioxane, chloroform, ethylacetate, acetonitrile, pyridine, dimethylformamide, and the like) in thepresence of 1-5 molar equivalents of an organic base (especiallyaromatic amines such as pyridine, trialkylamines, orN,N-dialkylanilines) (adapted from Fuji et al., U.S. Pat. No.4,425,335). The acyl substituent on the secondary amine can be the sameor different from those on the hydroxyl groups of the ribose moiety.

Example 5 Preparation of 3′,5′-Diacyl-2′-deoxycytidine

2-Deoxycytidine hydrochloride is dissolved in N,N-dimethylformamide. 2.1molar equivalents of the acid chloride of the desired acyl substituentis added, and the mixture is stirred overnight at room temperature. Thereaction mixture is concentrated in vacuo to an oil, and triturated witha mixture of ethyl acetate and diethyl ether or similar solvents. Theoil is then triturated with 1N sodium hydrogen carbonate. Thecrystalline solid is collected, washed with water, dried, andrecrystallized (adapted from Gish et al., J. Med. Chem. 14:1159 (1971)).

Example 6 Preparation of 5′-Acyl-2′-deoxycytidine

2-Deoxycytidine hydrochloride is dissolved in N,N-dimethylformamide. 1.1molar equivalents of the acid chloride of the desired acyl substituentis added, and the mixture is stirred overnight at room temperature. Thereaction mixture is concentrated in vacuo to an oil, and triturated witha mixture of ethyl acetate and diethyl ether or similar solvents. Theoil is then triturated with 1N sodium hydrogen carbonate. Thecrystalline solid is collected, washed with water, dried, andrecrystallized (adapted from Gish et al.).

Example 7 Preparation of N⁴-Acyl-2′-deoxycytidine

The N⁴-amino group of 2′-deoxycytidine is the best nucleophile among theamino and hydroxyl functionalities of deoxycytidine. SelectiveN⁴-acylation can be accomplished by treating 2′-deoxycytidine withappropriate acid anhydrides in pyridine or a mixture of pyridine andN,N-dimethylformamide. Specifically, deoxycytidine is suspended inpyridine, 1.5 molar equivalents of desired acid anhydride is added, andthe mixture is refluxed for about 2 hours. The solvent is removed invacuo, and the resulting white solid is recrystallized.

Alternatively, 2′-deoxycytidine is dissolved in 70:30pyridine:n,N-dimethylformamide. 1.5 molar equivalents of the acidanhydride of the desired acyl substituent is added, and the mixture isstirred overnight at room temperature, after which it is poured intowater and stirred. The solvent is removed in vacuo to leave a whitesolid, which is recrystallized (adapted from Sasaki et al., Chem. Pharm.Bul. 15:894 (1967)).

An alternative procedure is to dissolve 2′-deoxycytidine in a mixture ofwater and a water-miscible organic solvent (such as dioxane, acetone,acetonitrile, N,N-dimethylformamide, dimethylsulfoxide, tetrahydrofuran,etc.) and to treat that solution with about a twofold excess of anappropriate acid anhydride. For example, 1 gram of 2′-deoxycytidine,dissolved in 5 ml of water, is mixed with 15 to 100 ml dioxane (moredioxane is needed for more lipophilic substituents), and 2 molarequivalents of the acid anhydride of the desired acyl substituent isadded. The mixture is stirred for 5 hours at 80° C. (or for 48 hours atroom temperature), and then the solvent is removed in vacuo. The residueis washed with hexane or benzene, and recrystallized (adapted fromAkiyama et al., Chem. Pharm. Bull. 26:981 (1978)).

Example 8 Preparation of 3′,5′N⁴-Triacyl-2′-deoxycytidine

Compounds in which the acyl substituent of the N⁴ amino group and thehydroxyl groups of the ribose ring of deoxycytidine are the same (e.g.,triacetyl deoxycytidine) are prepared by dissolving or suspending2′-deoxycytidine in dry pyridine, adding at least 3 molar equivalents ofthe acid chloride or acid anhydride of the desired substituent, andstirring the mixture overnight at room temperature. The solvent isremoved in vacuo and the residue is washed and recrystallized.

Example 9 Preparation of 3′,5′-Diacyl-2′-deoxyadenosine

2′-Deoxyadenosine hydrochloride is dissolved in N,N-dimethylformamide.2.1 molar equivalents of the acid chloride of the desired acylsubstituent is added, and the mixture is stirred overnight at roomtemperature. The reaction mixture is concentrated in vacuo to an oil,and triturated with a mixture of ethyl acetate and diethyl ether orsimilar solvents. The oil is then triturated with 1N sodium hydrogencarbonate. The crystalline solid is collected, washed with water, dried,and recrystallized (adapted from Gish et al.).

Example 10 Preparation of 5′-Acyl-2′-deoxyadenosine

2′-Deoxyadenosine hydrochloride is dissolved in N,N-dimethylformamide.1.1 molar equivalents of the acid chloride of the desired acylsubstituent is added, and the mixture is stirred overnight at roomtemperature. The reaction mixture is concentrated in vacuo to an oil,and triturated with a mixture of ethyl acetate and diethyl ether orsimilar solvents. The oil is then triturated with 1N sodium hydrogencarbonate. The crystalline solid is collected, washed with water, dried,and recrystallized (adapted from Gish et al.).

Example 11 Preparation of N⁶-Acyl-2′-deoxyadenosine

The N⁶-amino group of deoxyadenosine is the best nucleophile among theamino and hydroxyl functionalities of deoxyadenosine. SelectiveN⁶-acylation can be accomplished by treatnig 2′-deoxyadenosine withappropriate acid anhydrides in pyridine or a mixture of pyridine andN,N-dimethylformamide. Specifically, deoxyadenosine is suspended inpyridine, 1.5 molar equivalents of desired acid anhydride is added, andthe mixture is refluxed for about 2 hours. The solvent is removed invacuo, and the resulting white solid is recrystallized.

Alternatively, 2′-deoxyadenosine is dissolved in 70:30pyridine:n,N-dimethylformamide. 1.5 molar equivalents of the acidanhydride of the desired acyl substituent is added, and the mixture isstirred overnight at room temperature, after which it is poured intowater and stirred. The solvent is removed in vacuo to leave a whitesolid, which is recrystallized (adapted from Sasaki et al., Chem. Pharm.Bul. 15:894 (1967)).

An alternative procedure is to dissolve 2′-deoxyadenosine in a mixtureof water and a water-miscible organic solvent (such as dioxane, acetone,acetonitrile, N,N-dimethylformamide, dimethylsulfoxide, tetrahydrofuran,etc.) and to treat that solution with about a twofold excess of anappropriate acid anhydride. For example, 1 gram of 2′-deoxyadenosine,dissolved in 5 ml of water, is mixed with 15 to 100 ml dioxane (moredioxane is needed for more lipophilic substituents), and 2 molarequivalents of the acid anhydride of the desired acyl substituent isadded. The mixture is stirred for 5 hours at 80° C. (or for 48 hours atroom temperature), and then the solvent is removed in vacuo. The residueis washed with hexane or benzene, and recrystallized (adapted fromAkiyama et al.).

Example 12 Preparation of 3′,5′N⁶-Triacyl-2′-deoxyadenosine

Compounds in which the acyl substituent of the N⁶ amino group and thehydroxyl group of the ribose ring of deoxyadenosine are the same (e.g.,tretraacetyl deoxyadenosine) are prepared by dissolving or suspending2′-deoxyadenosine in dry pyridine, adding at least 4 molar equivalentsof the acid chloride or acid anhydride of the desired substituent, andstirring the mixture overnight at room temperature. The solvent isremoved in vacuo and their residue is washed and recrystallized.

Example 13 Preparation of 3′,5′-Diacyl-2′-deoxyguanosine

2′-Deoxyguanosine hydrochloride is dissolved in N,N-dimethylformamide.2.1 molar equivalents of the acid chloride of the desired acylsubstituent is added, and the mixture is stirred overnight at roomtemperature. The reaction mixture is concentrated in vacuo to an oil,and triturated with a mixture of ethyl acetate and diethyl ether orsimilar solvents. The oil is then triturated with 1N sodium hydrogencarbonate. The crystalline solid is collected, washed with water, dried,and recrystallized (adapted from Gish et al.).

Example 14 Preparation of 5′-Acyl-2′-deoxyguanosine

2′-Deoxyguanosine hydrochloride is dissolved in N,N-dimethylformamide.1.1 molar equivalents of the acid chloride of the desired acylsubstituent is added, and the mixture is stirred overnight at roomtemperature. The reaction mixture is concentrated in vacuo to an oil,and triturated with a mixture of ethyl acetate and diethyl ether orsimilar solvents. The oil is then triturated with 1N sodium hydrogencarbonate. The crystalline solid is collected, washed with water, dried,and recrystallized (adapted from Gish et al.).

Example 15 Preparation of N²-Acyl-2′-deoxyguanosine

The N²-amino group of deoxyguanosine is the best nucleophile among theamino and hydroxyl functionalities of deoxyguanosine. SelectiveN²-acylation can be accomplished by treatnig 2′-deoxyguanosine withappropriate acid anhydrides in pyridine or a mixture of pyridine andN,N-dimethylformamide. Specifically, deoxyguanosine is suspended inpyridine, 1.5 molar equivalents of desired acid anhydride is added, andthe mixture is refluxed for about 2 hours. The solvent is removed invacuo, and the resulting white solid is recrystallized.

Alternatively, 2′-deoxyguanosine is dissolved in 70:30pyridine:n,N-dimethylformamide. 1.5 molar equivalents of the acidanhydride of the desired acyl substituent is added, and the mixture isstirred overnight at room temperature, after which it is poured intowater and stirred. The solvent is removed in vacuo to leave a whitesolid, which is recrystallized (adapted from Sasaki et al.).

An alternative procedure is to dissolve 2′-deoxyadenosine in a mixtureof water and a water-miscible organic solvent (such as dioxane, acetone,acetonitrile, N,N-dimethylformamide, dimethylsulfoxide, tetrahydrofuran,etc.) and to treat that solution with about a twofold excess of anappropriate acid anhydride. For example, 1 gram of 2′-deoxyadenosine,dissolved in 5 ml of water, is mixed with 15 to 100 ml dioxane (moredioxane is needed for more lipophilic substituents), and 2 molarequivalents of the acid anhydride of the desired acyl substituent isadded. The mixture is stirred for 5 hours at 80° C. (or for 48 hours atroom temperature), and then the solvent is removed in vacuo. The residueis washed with hexane or benzene, and recrystallized (adapted fromAkiyama et al.).

Example 16 Preparation of 3′,5′N²-Triacyl-2′-deoxyguanosine

Compounds in which the acyl substituent of the N² amino group and thehydroxyl groups of the ribose ring of deoxyguanosine are the same (e.g.,tretraacetyl deoxyguanosine) are prepared by dissolving or suspending2′-deoxyguanosine in dry pyridine, adding at least 4 molar equivalentsof the acid chloride or acid anhydride of the desired substituent, andstirring the mixture overnight at room temperature. The solvent isremoved in vacuo and their residue is washed and recrystallized.

Examples of Clinical Administration Radiation Exposure

Three situations wherein acyl derivatives of deoxyribonucleosides may beclinically useful in treating radiation damage are 1) accidentalexposure to ionizing radiation, as in a nuclear accident; 2) exposure toX-radiation during radiography; and 3) radiotherapy of cancer.

In the first case, acyl deoxyribonucleoside derivatives should beadministered in a formulation suitable for parenteral injection,followed by oral administration several times per day of dosesequivalent to 0.5 to 2 grams of each of the four majordeoxyribonucleosides. It is essential that the derivatives of all of thenucleosides be coadministered.

In the second case, X-ray exposure during diagnostic radiography, acyldeoxyribonucleside derivatives are given orally before and afterexposure.

In the third case, during cancer radiotherapy, the acyl ribonucleosidederivatives are particularly useful in restoring bone marrow functionafter its undesirable but unavoidable suppression during irradiation.Moreover, in formulations designed to selectively deliver nucleosides tonormal but not neoplastic tissues, the acyl nucleoside derivatives willimprove the therapeutic index (ratio of efficacy to toxicity) of theradiation treatment.

Wound Healing

In promoting the healing of skin wounds (whether surgical incisions oraccidental wounds), it is best to apply acyl deoxyribonucleosidederivatives topically, either in an ointment or in bioerodiblemicrocapsules. A topical antibiotic might be coadministered. The molarequivalent of 2 to 20 mg of a mixture of all four majordeoxyribonucleosides should be applied per square cm of wound area, or 1to 10 mg per cm of linear incision. The onset of the earliest phases ofwound healing in particular is accelerated.

Liver Regneration

Acyl derivatives of deoxyribonucleosides are useful in promotingregeneration of damaged or diseased liver, particularly for acceleratingregrowth after surgical removal of a portion of the liver. In this case,oral administration of the derivatives is preferable, in dosescorresponding to the molar equivalents of 0.2 to 2 grams of eachnucleoside. It is important that derivatives of all four majordeoxyribonucleosides be coadministered.

What is claimed is:
 1. A method of enhancing the delivery of exogenousdeoxyribonucleosides to the tissue of an animal, comprising the step ofadministering to said animal an effective amount of an acyl derivativeof 2′-deoxyadenosine, having the formula

wherein R₁, R₂, and R₃ are the same or different and each is hydrogen oran acyl group derived from (a) an unbranched fatty acid with 3 to 22carbon atoms, (b) an amino acid selected from the group consisting ofglycine, the L forms of alanine, valine, leucine, isoleucine, tyrosine,proline, hydroxyproline, serine, threonine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, carnitine, and ornithine,(c) nicotinic acid, or (d) a dicarboxylic acid having 3 to 22 carbonatoms, provided that not all of R₁, R₂, and R₃ are H, and where R₃ isnot H, then R₁ and/or R₂ may also be acetyl, or a pharmaceuticallyacceptable salt thereof.
 2. A method of enhancing the delivery ofexogenous deoxyribonucleosides to the tissue of an animal, comprisingthe step of administering to said animal an effective amount of an acylderivative of 2′-deoxyguanosine having the formula

wherein R₁, R₂, and R₃ are the same or different and each is hydrogen oran acyl group derived from (a) an unbranched fatty acid with 3 to 22carbon atoms, (b) an amino acid selected from the group consisting ofglycine, the L forms of alanine, valine, leucine, isoleucine, tyrosine,proline, hydroxyproline, serine, threonine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, phenylalanine, carnitine,and omithine, (c) nicotinic acid, or (d) a dicarboxylic acid having 3 to22 carbon atoms, provided that not all of R₁, R₂, and R₃ are H, andwhere R₃ is not H, then R₁ and/or R₂ may also be acetyl, or apharmaceutically acceptable salt thereof.
 3. A method of enhancing thedelivery of exogenous deoxyribonucleosides to the tissue of an animal,comprising the step of administering to said animal an effective amountof an acyl derivative of 2′-deoxycytidine, having the formula

wherein R₁, R₂, and R₃ are the same or different and each is hydrogen oran acyl group derived from (a) an unbranched fatty acid with 3 to 22carbon atoms, (b) an amino acid selected from the group consisting ofglycine, the L forms of alanine, valine, leucine, isoleucine, tyrosine,proline, hydroxyproline, serine, threonine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, carnitine, and ornithine,(c) nicotinic acid, or (d) a dicarboxylic acid having 3 to 22 carbonatoms, provided that not all of R₁, R₂, and R₃ are H, and where R₃ isnot H, then R₁ and/or R₂ may also be acetyl, or a pharmaceuticallyacceptable salt thereof.
 4. A method of enhancing the delivery ofexogenous deoxyribonucleosides to the tissue of an animal, comprisingthe step of administering to said animal an effective amount of an acylderivative of 2′-deoxythymidine, having the formula

wherein R₁ is an acyl group derived from (a) an unbranched fatty acidwith 3 to 15 or 17 to 22 carbon atoms, (b) an amino acid selected fromthe group consisting of glycine, the L forms of alanine, valine,leucine, isoleucine, tyrosine, proline, hydroxyproline, serine,threonine, cysteine, aspartic acid, glutamic acid, arginine, lysine,histidine, camitine, and ornithine, (c) nicotinic acid, or (d) adicarboxylic acid having 3 to 22 carbon atoms, and R₂ and R₃ are H, or apharmaceutically acceptable salt thereof.
 5. A method of enhancing thedelivery of exogenus deoxyribonucleosides to the tissue of an animal,comprising the step of administering to said animal an effective amountof an acyl derivative of 2′-deoxythymidine, having the formula

wherein R₁ is H, R₂ is an acyl group derived from (a) an unbranchedfatty acid with 3 to 13 or 15 to 22 carbon atoms, (b) an amino acidselected from the group consisting of glycine, the L forms of alanine,valine, leucine, isoleucine, tyrosine, proline, hydroxyproline, serine,threonine, cysteine, aspartic acid, glutamic acid, arginine, lysine,histidine, carnitine, and ornithine (c) nicotinic acid, or (d) adicarboxylic acid with 3 to 22 carbon atoms, and R₃ is H or apharmaceutically acceptable salt thereof.
 6. A method of enhancing thedelivery of exogenous deoxyribonucleosides to the tissue of an animal,comprising the step of administering to said animal an effective amountof an acyl derivative of 2′-deoxythymidine, having the formula

wherein R₁ and R₂ are the same or different and each is an acyl groupderived from (a) an unbranched fatty acid with 5 to 22 carbon atoms, (b)an amino acid selected from the group consisting of glycine, the L formsof alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cysteine, aspartic acid, glutamicacid, arginine, lysine, histidine, camitine, and ornithine, (c)nicotinic acid, or (d) a dicarboxylic acid with 3 to 22 carbon atoms,and R₃ is H or a pharmaceutically acceptable salt thereof.
 7. A methodof enhancing the delivery of exogenous deoxyribonucleosides to thetissue of an animal, comprising the step of administering to said animalan effective amount of an acyl derivative of 2′-deoxythymidine, havingthe formula

wherein R₁, and R₂ are the same or different and each is an acyl groupderived from (a) an unbranched fatty acid with 2 to 22 carbon atoms, (b)an amino acid selected from the group consisting of glycine, the L formsof alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cysteine, aspartic acid, glutamicacid, arginine, lysine, histidine, carnitine, and ornithine, (c)nicotinic acid or (d) a dicarboxylic acid with 3 to 22 carbon atoms, andR₃ is an acyl group derived from an optionally substituted benzoyl orheterocyclic carboxylic acid that is substantially nontoxic, or apharmaceutically acceptable salt thereof.
 8. A method of enhancing thedelivery of exogenous deoxyribonucleosides to the tissue of an animal,comprising the step of administering to said animal an effective amountof each of at least two compounds selected from at least two of thegroups of compounds having formulae

wherein R₁, R₂, and R₃ are the same or different and each is H or anacyl group derived from a carboxylic acid, provided that at least one ofsaid substituents R₁, R₂, and R₃ groups of compounds is not hydrogen, orpharmaceutically acceptable salts thereof.